The neuronal architecture of the anteroventral cochlear nucleus of the cat in the region of the cochlear nerve root: Horseradish peroxidase labelling of identified cell types

Neurostirnce

030&4522/82/l

Vol. 7. pp. 3031 to 3052, 1982

Printed in GreatBritain

23031-22603.00/O

Pergamon Press Ltd IBRO

THE NEURONAL ARCHITECTURE OF THE ANTEROVENTRAL COCHLEAR NUCLEUS OF THE CAT IN THE REGION OF THE COCHLEAR NERVE ROOT: HORSERADISH PEROXIDASE LABELLING OF IDENTIFIED CELL TYPES L. P. TOLBERT*, D. K. MOREST~ and D. A. YURGELUN-TODDY *,t,$Department of Anatomy, Harvard Medical School, Boston, MA 021 IS, U.S.A. and TDepartment of Anatomy, The University of Connecticut Health Center, Farmington, CT 06032. U.S.A. Abstract-Golgi

impregnations of the posterior part of the cat’s anteroventral cochlear nucleus have revealed two types of neurons, bushy cells with short bush-like dendrites and stellate cells with long, tapered processes: Nissl stains have revealed globular and multipolar cell bodies with dispersed and clumped ribosomal patterns, respectively. In the present study, we injected horseradish peroxidase into the trapezoid body. Ipsilaterally. retrograde, diffuse labelling of neurons, presumably through damaged fibers, yielded Golgi-like profiles of numerous bushy cells with typical dendrites and with thick axons projecting toward the trapezoid body. Stellate cells were almost never labelled in this way. Anterograde diffuse labelling of thick axons demonstrated calyx endings in the contralateral medial nucleus of the trapezoid body. In the electron-microscope, the perikarya of diffusely-filled bushy neurons were found to have the dispersed ribosomal pattern and the kinds of synaptic endings typical of globular cells, including large profiles of end-bulbs from cochlear nerve axons. After injections restricted to the medial trapezoid nucleus, granularly-labelled cells in the cochlear nucleus were almost completefy confined to the contralateral side: Nissl counterstaining showed them to be globular cells in the posterior part of the anteroventral cochlear nucleus. After larger injections, involving surrounding regions of the superior olivary complex, granular labelling occurred throughout the ventral cochlear nucleus on both sides. There is also evidence that stellate cells in Golgi impregnations correspond to multipolar cell bodies in Nissl stains. We conclude that bushy cells typically correspond to globular cells. which receive end-bulbs from the cochlea and send thick axons to the contralateral medial trapezoid nucleus, where they form calyces on principal cells. Principal cells, in turn, are known to project to the lateral superior olive and to one of the nuclei of origin of the crossed olivo-cochlear bundle, which feeds back to the cochlea. In this circuit, correlations between synaptic patterns and particular physiological signal transfer characteristics can be suggested. These could be related to binaural intensity interactions in the lateral superior olive and to a regulatory loop involving the olivo-cochlear bundles.

The cochlear nucleus is an intricate and highly ordered neuronal web. Yet, despite its complexity as a whole, it can be divided into discrete subnuclei, each of which contains relatively few morphological cell types and exhibits correspondingly few physiological response patterns. The posterior division of the anteroventral cochlear nucleus (AVCNp) of the cat contains globular and multipolar cells, as defined with the Nissl stain and identified in electron-micrographs,

* Present address: Department of Anatomy, Georgetown University School of Medicine, 3900 Reservoir Rd., Washington, D.C. 20007, U.S.A. t Send reprint requests to: Dr D. Kent Morest, Department of Anatomy, University of Connecticut Health Center, Farmington, CT 06032, U.S.A. Ahbreoiations: AVCN, anteroventral cochlear nucleus; AVCNp, posterior division of AVCN: HRP, horseradish peroxidase; MNTB, medial nucleus of the trapezoid body; PD, dorsal part of AVCNp; PV, ventral part of AVCNp.

and two basic neuronal types, the bushy and stellate neurons, as defined with the Golgi methods.44+45 On the basis of size, shape, number of dendrites and distribution within AVCNp, one can postulate correspondences between the neuronal types defined with the different methods. Globular cells, receiving large cochlear nerve end-bulbs, should correspond to bushy neurons, which have short tufted dendrites; multipolar cells, receiving few axosomatic synapses, should correspond to stellate neurons, which have long tapered dendrites. 45 Moreover, because some endbulbs appear to produce a recognizable wave form in electrophysiologi~al recordings,’ the globular cells can be matched tentatively with the category of unit responses resembling those of the cochlear nerve. Stellate cells may correspond to other unit types. The present study demonstrates direct correlations between bushy neurons and globular cells by using electron-microscopy of neurons diffusely-filled with horseradish peroxidase. 3031

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t. P. Toibert. D. K. Merest and D. A. Yurgelun-Todd

Cells in the region of the cochlear nerve root contribute a large-fiber component to the trapezoid body, which forms the large axosomatic catyces of Held on the principal neurons in the medial nucleus of the trapezoid body (MNTB). i5~‘6~30~48 The principal cells respond to simple stimuli in much the same way as globular cells are supposed to.22 However, the identity of the cells forming the calycine axons remains to be demonstrated by direct methods. The present study uses a combined anterograde-retrograde transport method to establish this identity.

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PROCEDURES

Nine healthy cats, one adult and eight 4-t/2-6-t/2 months-old, were used. Under pentobarbital anesthesia, the trachea was cannulated and the basi-occipital bone, exposed and opened. fn two cats (Cl and C2), a longitudinal cut was made in the trapezoid body 2mm deep and 3 mm long, just lateral to the right pyramid and the abdutens nerve root. with a scalpel (No. 11 Bard-Parker). In these incisions, large injections (3-4 pl) of 3Ou/:,horseradish peroxidase (HRP) (Sigma Type VI or Worthington) in normal saline were delivered in multiple spurts by manual pressure through glass pipettes (approximately 40nm tip diameter). In the 6 other cats, the pipettes, hand-held or mounted on a micromanipu~ator, were lowered approximately 1.5 mm inro the intact trapezoid body. In two of these cases (C3 and C4), injections were delivered just as for the transected trapezoid bodies. In the remaining 4 cases (C5-C8). smaller injections (0.10~0.15 /II) were delivered over 15 min with a syringe microdrive. The pipette was left in place for 10 min before removal. Following surgery. cats were maintained for 18-44 h under light anesthesia. The extent of the injection and the survival time for each cat are shown in Table I. At the end of the experiment+ each cat was perfused through the heart with about SOml of warm isotonic saline followed by a warm fixative containing 0.51,; paraformaldehyde and 2.54,, giutaraidehyde (freshly cleaned with charcoal) in a 0.1 M phosphate buffer with 0.008’~~calcium chIoride.s’ Following perfusion. the animal was decapitated and enough tissue and skull were removed From the head to ensure exposure of the brain to fixative. The head was immersed in fixative for 6-24 h in the cold. The brain stem was then removed from the skull and cut into 1 cm lengths, which were immersed in XI?;, buffered sucrose until they just started to sink. The blocks were frozen with COZ and cut at 4@-50gm. Some sections for both light- and electron-microscopy were treated for 15-30 min in O.S-1.02, cobaltous chloride” in TRIS buffer, and then all sections were reacted with diaminobenzidine’ * in phosphate buffer. pH 7.4. Pretreatment with CoCI, blackened the HRP reaction product but did nor change its appearance in the electron-microscopic Sections were examined under glycerine and coverglasses with the fight-microscope and cells chosen for electronmicroscopy were quickly sketched. As soon as possible, sections containing these cells were post-fixed in osmium tetroxide, stained with uranyt acetate, and embedded in Epon resin. Under light-microscopic control each block was trimmed down to the filled cell. For light-microscopy some, usually alternate, sections were counterstained with Cresyl Violet or thionin. Magnifi-

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Horseradish peroxidase in cochlear nucleus cations of 40 x or 100 x were used to identify HRP granules in lightly-labelled cells, with or without dark-field illumination. In each cat, the locations of injection sites, defined as the regions containing diffuse extra-neuronal reaction product, and of ail labelled cells in approximately every tenth section were mapped with the aid of a drawing attachment. Likewise, the outlines of 119 diffusely-filled cells were drawn for measurements. Available for comparison was a series of Golgi impregnations from the auditory nuclei of fully adult cats.44 As a control, in one cat (not in Table 1). HRP was injected into the pyramid of one side 24 h before death. Granules of HRP reaction product were not detectable in neurons in any of the auditory nuclei, even though the enzyme reaction appeared in the pyramid and in red blood cells in the auditory nuclei.

RESULTS Injection

sites

In the present study, the injection site is defined by two concentric contours. The inner contour outlines the region where the granular reaction product is so dense that cellular outlines cannot be resolved; the outer contour encompasses the zone of diffuse, moderate-to-pale, extra-neuronal HRP. These two zones represent the most liberal and the most conservative definitions of uptake sites (Figs l-4). The injections center on the MNTB and include varying amounts of the superior olivary complex of one side (Table 1). In cats Cl, C2 (Fig. 11,and C5 (Fig. 2), the injection site spares most of the lateral superior olive but includes the medial superior olive and MNTB; faint reaction product spreads laterally to the fiber capsule of the lateral superior olive and medially along the trapezoid body just across the midline. In cats Cl and C2, the trapezoid body is transected just medial to the abducens nerve root. In cats C3 and C4, the injection site spans all of one olivary complex, the MNTB. and smaller peri-olivary nuclei; diffuse HRP also encroaches on the reticular formation dorsally, the pyramidal tract ventrally, and the midline medially. The injection in cat C6 (Fig. 3) is confined to the MNTB and to a distinct band in the medial superior olive through its posterior two-thirds; this band corresponds approximately to the 1-4 kHz frequency representation.’ 3 In cats C7 and C8 (Fig. 4), the injection site is limited to the MNTB and the area of trapezoid body fibers immediately medial and ventral to it on one side. Neuronal labelling Two types of labelling characterize the present findings: the diffusely-filled neurons and axons and the granularly-labelled cell bodies. The diffusely-filled cells contain a continuous field of reaction product and resemble Golgi imprecations. Granular labelling consists of discrete intracellular granules of reaction product visible in the perikaryal cytoplasm and sometimes in dendritic trunks. In this study, primary reliance is on granularly labelled cells in defining ori-

3033

gins of projections. Diffusely-filled neurons are used to study the light- and electron-microscopic characteristics of identified cell types; they yield information about the paths of axons but do not necessarily demonstrate their destinations. ~~~use~~~~l~edcells and axons look like Golgi impregnations Cell bodies ana’ dendrites. In five cats (C2, C3, C4, CS, and C6), with survival times of 3&44 h, many cells are diffusely filled in AVCNp ipsilaterally. Cats C5 and C6 have the most darkly and completely filled cells. In cat C7, the brain stem bulged into the craniotomy defect, and there was little granular or diffuse labelling, possibly because of compression of the trapezoid body. There are no diffusely-labelled cochlear nucleus neurons outside the ipsilateral AVCNp in these cats or anywhere in the cochlear nucleus of cats Cl and C8, which had the shortest survival times. However, in all cats the cochlear nuclei contain granularly labelled neurons (Table 1). The diffusely-filled cells were compared with adult Golgi material. All but three of the it9 diffusely-filled cells were identified as bushy cells by their dendritic morphology (Figs 5,6). The filled bushy neurons have 1-3 slender dendritic trunks, with many right-angle processes. The primary dendrites are 2-13Opm long, the bushy processes, up to 200 pm. The dendritic processes on some cells are thick and form a dense bush (Figs 6A-D), on some they are thin and form a sparse bush (Figs 6E, F), and on others many of the finer processes are varicose and form an extremely dense mesh near the cell body (Figs 6G, H). The oval cell body is often covered with spicules; it is 24/*m in average diameter, with a mean width-to-length ratio of 0.7. The nucleus, visible as a lighter zone, is usually eccentric. As the axon leaves the soma, it is about 1 pm in diameter, but after some 30pm, it twists, sometimes with a constriction, and abruptly thickens to 2.53 pm in diameter (Fig. 6C). This sudden change in diameter characterizes all bushy cells from which the axons can be traced for more than 20pm. No branches appear on the initial portions of the bushy cell axons. The three exceptional cells in the diffusely-filled group are stellate cells in the PD of cat C6. One has a filled axon arising from the cell body, l-2.5 pm thick along its jagged course out of the cochlear nucleus; unlike the bushy cell axons, it forms a collateral in the cochlear nucleus (Fig. 7). Axons. Many axons in the trapezoid body and ipsilateral cochlear nucleus are diffusely filled in both the retrograde and anterograde direction, From the injection site, filled fibers extend in the trapezoid body laterally, into the ipsilateral superior olivary complex and cochlear nucleus, and medially, across the midline into the superior olivary complex and toward the opposite cochlear nucleus. The filled axons that cross the midline are primarily large caliber fibers (253.0pm in diameter), many of which end as

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L. P. Tolbert,

D. K. Morest

calyces in the contralateral MNTB (Fig. 8). The calyces are generally similar in appearance to those described with the Golgi method in kittens3’ and adult cats (not shown). They consist of finger-like processes, up to 30pm long, grasping a cell body: protruding from these processes are numerous appendages Iike those in Golgi impregnations. Calycine collaterals are not seen: however. they can be found in adult Golgi impregnations, where they may be less prevalent and less extensive than in kittens. Axons corresponding in distribution and appearance to precalycine collaterals are visible, but. running out of the plane of section, they cannot be traced individually from calyciferous fibers. Beyond the MNTB, contralaterally. there are few filled fibers; these generally disappear at or near the medial border of the cochlear nucleus. Ipsilaterally, the trapezoid body contains filled axons ranging from less than 1 to over 3 pm in diameter. Individual axons cannot be followed from the injection site into the cochlear nucleus in single transverse sections, but axon segments of the same diameters cover the entire course. The thickest axons form thin collaterats beneath the lateral superior olive (Fig. 9). These collaterals run in three fascicles to the lateral nucleus of the trapezoid body, where they arborize in a dense plexus. In ipsilateral MNTB at the edge of an injection site, the thick axons form heavily filled calyces on neurons which have a light, diffuse coloration that reveals dendrites like the primary dendrites of principal cells seen with Golgi methods. in the ipsilateral cochlear nucleus, most filled axons cross the medial border of AVCNp. Some. the thickest, can be traced from the bodies of diffusely-filled bushy cells, but many thinner axons extend horizontally across PD, parallel to the conspicuous fascicles which characterize this zone (Fig. 5). These thinner axons are not collaterals but appear to arise outside the cochlear nucleus; however, their terminal arborizations are not seen. Other filled axons course dorsally along the medial edge of the AVCN before curving laterally to enter the posterior part of the anterior division and to form clusters of small boutons throughout the anterior division and also a few in PD. PV, and the dorsal cochlear nucleus.

and D. A. Yurgelun-Todd

Direct evidence that bushy cells receive end-bulbs Seven diffusely-filled bushy cells, clearly identified in the light-microscope (5 from PV and 2 from PD), were examined in the electron-microscope. Aside from holes due to ice crystals, the membranes and many cytoplasmic organelles are intact. A grainy electrondense reaction product fills the cell body and its appendages (Fig. 10). In every case. the bushy neurons can be identified as globular cells. Rough endoplasmic reticulum is scattered or gathered in small stacks, only 3-4 cisterns deep. The plasma membrane is wavy, conforming to the shapes of the synaptic terminals. and forms somatic spicules and longer appendages (Fig. 10). Synaptic terminals cover the profile of the cell. These terminals are of the three types previously described45--type 1. including end-bulbs, with large round vesicles (Fig. 10. I). type 2 with small round-to-oval vesicles (Fig. 10, 2), and type 3 with flattened vesicles. In the neuropil near the filled cell bodies are the profiles of filled dendrites, all, or almost all of which must belong to bushy cells in the same or nearby sections, for the stellate cell dendrites so rarely fill. These dendritic profiles range from several microns to less than 0.5 pm thick, and they receive synaptic terminals only rarely (Fig. 13). Axonal projections of neurons in posterior division of anteroventral cochleur nucleus As shown in Table 1. granularly-labelled cells occur throughout the ventral cochlear nllcieus on both sides in all cats, except C7 and C8, where the labelling is essentially confined to neurons of the contralateral AVCNp. A few labelled cells also appear in the dorsal cochlear nucleus, the nuclei of the lateral lemniscus, and the inferior colliculus. In Cl and C2 (Fig. I), in which the trapezoid body was partially severed, many cells throughout the ventral cochlear nucleus of both sides contain HRP granules. The cells range from lightly-labelied to almost completely-filled. In counterstained sections from C2, the lightly-labelled cells are primarily spherical cells in the anterior AVCN or globular ones in AVCNp.

Fig. I. Location of injection site and of labelled neuronal cell bodies in the cochlear nucleus of cat C2, in every f&b section, rostralmost at top left. caudalmost at bottom right. Arrows, site of trapezoid body transection. 5-month-old cat, 33 h survival, 50pm thick sections. Scale = 2 mm. In this and subsequent figures, the injection site is indicated by two concentric areas of shading. In the central. black zone dense granular HRP reaction product obscures cellular outlines. The outer. gray region includes all of the diffuse. extra-neuronal reaction product. Dots in the cochlear nuclei and inferior colliculus indicate the positions of labelled cells. The relative numbers of dots reflect the relative numbers of labelled cells. Abbreviations for this and succeeding figures: AA. anterior part of AVCNa (anterior division of anteroventral cochlear nucleus); AP. posterior part of AVCNa; APD, posterodorsal part of AVCNa; DCN, dorsal cochlear nucleus; CCL, granule cell layer; IC, inferior colliculus; LSO, lateral superior olive; MNTB, medial nucleus of trapezoid body; MSO, medial superior olive; PD, dorsal part of AVCNp (posterior division of anteroventral cochlear nucleus); PV. ventral part of AVtNp: PVCN, posteroventral cochlear nucleus; pyr. tr., pyramidal tract; TB, trapezoid body; VNLL, ventral nucleus of lateral lemnisctts; t.V. descending trigeminal tract; VI, abducens nerve root; VII, nVlI. facial nerve root and nucleus; VlIIv, nVIlIv, vestibular nerve root and nucleus.

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Horseradish peroxidase in cochlear nucleus The most heavily labelled cells are stellate cells that rim the AVCN (Fig. 11; including the ‘small cell cap’ of Osen33). A few lightly-labelled cell bodies are also seen in the ipsilateral deep dorsal cochlear nucleus, the contralateral nucleus of the intermediate acoustic stria, and in the contralateral nucleus of the lateral lemniscus. When the injection site involves all of the superior olivary complex of one side (C3, C4), most cells are labelled in the ventral cochlear nucleus on both sides. In these cases, a few labelled cells occur in the ipsilateral central nucleus of the inferior colliculus and, in one cat (C3), in the nucleus of the intermediate acoustic stria and the fusiform cell layer of the dorsal cochtear nucleus. In all three subdivisions of the anterior AVCN, there are many unlabelled ovoid cell bodies (terminology of Brawer & Morests), but few, if any, unlabelled spherical cell bodies, In the AVCNp, most, if not all, of the globular cell bodies and a smaller number of multipolar cell bodies are labelled. Cats C5 and C6 have injection sites that include the MNTB and part of the medial superior olive, with possible involvement of the ventral peri-olivary cell groups. Labelled cells are restricted to the ventral cochlear nucleus (Figs 2, 3). In AVCNp, almost all globular. and very few multipolar cell bodies are labelled. In C5, all of the cells in PV and ventral PD contralateral to the injection were counted in two sections: 259 out of 285 globular, and 13 out of 135 multipolar cell bodies labelled. In C5, where the injection involves most of the medial superior olive, most of the spherical cells in anterior AVCN on both sides are lightly labelled. In contrast, in C6, where a band through the medial superior olive was injected, the anterior AVCN differs on the two sides: contralaterally, there is a band of heavily-labelled spherical cells (Fig. 3) with a few lightly labelled spherical cell bodies elsewhere; ipsilaterally, there are few labelled ceils, with none in the anterior and posterodorsal parts. The band of heavily-labelled cells in AVCN centers on the 3 kHz plane.4 This correlates with the l-4 kHz value estimated (after Guinan et ~1.‘~) for the band in the medial superior olive. In C7 and C8, the injection site was restricted to the MNTB, excluding its anterior and posterior poles. Labelling was largely confined to globular cell bodies in the contralateral AVCNp, especially in the cell cluster in lateral PV (Fig. 12). No identifiable multipolar cell bodies contain HRP granules. In C7, despite the diminished transport of HRP, there are many lightly-labelled globular cells (Table 1). In C8, approximately one-half of the globular cell bodies in the contralateral AVCNp (Fig. 4), but hardly any ipsilaterally, are labelled. Electron-microscopy of granularly labelled cells

A few small, granularly labelled stellate cells, like those in Fig. 11, from the medial border of PD were examined in C2 by electron-microscopy. The cell bodies contain large arrays of rough endoplasmic reti-

culum and have very few synaptic terminals (Fig. 13). They clearly correspond to multipolar cell bodies.45 DISCUSSION The present study confirms the correlations previously suggested between bushy cells in Golgi impregnations and globular cell bodies in Nissl stains.“5 It shows that bushy cells send axons to the MNTB and form axosomatic calyces on the principal cells there. There is also evidence that stellate cells in Golgi impregnations correspond to multipolar cell bodies in Nissl stains. We discuss, first, some of the Iimitations of our methods and then the interpretations. Mode of ~orser~~s~ perox~dase uptake IX&se filling. Diffuse filling of neurons probably results from leakage of HRP across a damaged plasma membrane and subsequent migration through the axoplasm into the axonal endings and to the cell body and dendrites.18,25 Grunulur labelhy. Granular labelling can occur by endocytosis and by lysosomal sequestration in diffusely-filled cells or axons. Endocytotic granular labelling has been shown following uptake at synaptic terminals,23*2” and, to a lesser extent, along the shaft of the axon*9+24*25*29Lysosomal sequestration of diffuse HRP has also been described.9+‘4*24 Interpretation of experiments. In the present material, cells show various degrees of labelling, ranging from uniformly filled cells to a barely detectable sprinkling of granules. Perhaps more sensitive substrates could demonstrate more neurons. The degree of diffuse filling correlates with the extent of damage during injection, but the amount of granular labelling does not. We assume that diffuse filling occurs through damaged axons and that granular labelling is mostly from endocytosis by synaptic terminals and. to a lesser extent perhaps, by axons. bight-microscopy of diffusely-~lled

cells and axons

Cells in the cochlear nucfeus. Of the diffusely-filled cells in 2 experimental animals, 116 are bushy cells and 3 are stellate cells. The bushy cells closely resemble their Golgi counterparts from 1-l/2--4-month-old kittens. Apparently, the form attained by bushy cells at 54 days of age persists at least through 6 months and probably represents the mature form, in agreement with Tolbert & Morest.44 In the present study no collaterals of bushy cell axons appear in the cochIear nucleus or run back to it, in agreement with Brawer ei al.’ but not Lorente de No? The single stellate cell axon that could be followed in the present study does form a recurrent collateral. Brawer et aL6 also found recurrent collaterals of stellate cells in AVCN. Lorente de Noz6*” stated that ‘spherical’ and multipolar cells of AVCNp send collaterals into a ventrotubercular tract to the dorsal cochlear nucleus. Centrifugal axons. Fine axons enter the AVCN from the trapezoid body and form clusters of boutons.

L. P. Tolbert, D. K. Merest and D. A. Yurgelun-Todd

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The origins and terminations of these pathways’ are still not completely known in detail, although a number of studies suggest origins in the superior olivary complex and inferior coIliculus2~10~20~37~36~40 and in the nuclei of the lateral lemniscus.2’ In the present study, terminals in AVCNp presumably filled after interruption of centrifugal axons in the trapezoid body. These may correspond to one of the types of non-cochlear fibers discussed previously.44 Electron-microscope

of d$fuseiy Jilled cells

Cells filled with HRP were used for direct correlations between cell types. On the one hand, these were defined primarily on the basis of dendritic morphology, using the Golgi method. On the other hand, cell types were defined on the basis of intracellular distribution of rough endoplasmic reticulum, using the Nissl stain and electron-microscopy. It is unlikely that the diffusely-filled neurons examined in the electron-microscope were chromatolytic multipolar cell bodies. The arrangement of the rough endoplasmic reticulum in small stacks of cisterns, often near the plasmalemma, is identical to that of normal globular

cell bodies. Also, the diffusely-filled perikarya receive the multiple axosomatic synapses characteristic of globular cell bodies but not of multipolar cell bodies. Several types of bushy cells are distinguishable, but it remains for future studies to identify these in Nissl preparations. The present correlations establish a picture of the synaptic input to bushy cells and stellate cells, based on previous studies.45 This is summarized in Fig. 14. Bushy cell bodies, which have eccentric nuclei and a dispersed ribosomal pattern, are almost covered with synaptic endings. More than half of the endings, including the large end-bulbs, are from cochlear nerve fibers. The rest are from non-cochlear sources. Stellate and elongate cell bodies, which usually contain large stacks of rough endoplasmic reticulum, are almost devoid of synapses. Roughly equal numbers of cochiear and non-cochtear synapses cluster at the bases of their dendrites. Projections of the cochleur nucleus The results indicate that neurons in all AVCN subdivisions send their axons to or through the superior olivary complex of both sides. Injections involving the

Fig. 5. Diffusely-filled neurons in Nissl-counterstained transverse section through anteroventral cochlear nucleus from cat CS. One ceil body (a). the dendrite of which is not in this section, has an axon which, after a thin initial segment (*). expands to 3pm in diameter. Other neurons have tilled dendrites that identify them as bushy ceils (b). in PV. with thick fascicles of unlabelled cochlear nerve fibers, and in PD. with thin, filled axons (arrows). Granular labelling in Nissl-stained perikarya is not visible at this magni~cation. c, dendrites of a bushy cell whose body is in the adjacent section: D, dorsal; M, medial. Scale = 50 fern. Fig. 6. Diffusely-filled bushy cells in posterior division of anteroventral cochlear nucleus. A. C--H. from C6; B. from C5. C. the axon has a thin. tapering initial segment which undergoes a constriction (large arrowhead) before expanding to about 3 pm in diameter. D. on an elongated cell body the spicules stop abruptly at the base of a dendrite (open triangle). E. F. bushy cells with sparse dendritic branches at nearly right angles to the dendritic trunks: cell body is spiculated in E and smooth in F. G. H. bushy cells with dense arrays of fine varicose dendrites which often extend parallel to the cochlear nerve fibers (directions of arrows). g. granularly labelled globular cells, small arrowheads, somatic spicules. Transverse section. Scale = 25 itm. Fig. 7. Diffusely-tilled stellate cell (s) in Nissl-counterstained transverse section through anteroventral cochlear nucleus from cat C6. one of only three stellate cells filled in the present material. The stellate axon (a) projects toward the trapezoid body (TB) after emitting (arrow) a collateral (c): which does not form a bouton in this section. A diffusely-filled bushy cell (b. seen also in Fig. 6A) sits on the border between PD above. and PV below, with its slews of thick, filled axons coursing toward the trapezoid body. Thinner. filled axons form nests of endings on globular cell bodies (open triangles). D, Dorsal; L. Lateral. Scale = 50hfm. Fig. 8. Djffusely-filled calyces in transverse section from the medial trapezoid nucleus contral~iteral to the horseradish peroxidase injections in cats C6 (top) and C5 (bottom). Arrows. grape-like calycine appendages.

Fig. 9. Thick horizontal fibers of the trapezoid body (TB) forming vertical collaterals that arborize in the lateral trapezoid nucleus (LNTB) from cat C2. Thinner, unfilled fibers are visible in the trapezoid body. Scale = 50ifrn. Inset: Enlargement of the area within the rectangle. showing origin of the vertical collaterals (c) from thick trapezoid fibers. Scale = 50 om. Fig. 10. Electron-micrographic montage of part of a diffusely-filled bushy cell body in PV that was seen in the light-microscope to have knob-like appendages and bushy dendrites. Reaction product tills perikaryon and appendages (*); the soma receives many type I and 2 synaptic endings. Unstained section. Scale = l.Ojrm.

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Fig. 13.Electron-micro~raph montage of granularly labelled small horizontal stellate cell from PD in cat CL The nucleus (n) is centrally located in the small cell body. The cytoplasm contains a stack of rough endoplasmic reticulum (RER) and numerous dark lysosomes (ly) as well as very light diffuse stain. The few Impinging synapses are labelled ‘I’ or ‘3’ according to type. Scale = 5 jtm.

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Horseradish peroxidase in cochlear nucleus medial superior olive, MNTB, and medial peri-olivary nuclei of one side result in heavy labelling of cells in all of the AVCN subdivisions bilaterally; thus, these subdivisions appear to project bilaterally to that region. Injections limited to the MNTB alone label cells almost exclusively in the contralateral AVCNp. Diffuse labelling of cells and axons suggests that bushy cells in the AVCNp also send collaterals to the ipsilateral lateral trapezoid nucleus. 0sen34 suggested that the anterior part of the anterior AVCN projects to the medial superior olive bilaterally and that the posterior part of the anterior AVCN projects to the ipsilateral lateral superior olive. The present study indicates that many cells in the posterior, as well as the anterior, parts of the anterior AVCN project to the medial superior olive bilaterally. These results agree with the frequency maps for the medial superior olive13 and cochlear nucleus,4 which indicate a small, high frequency representation in the ventral tip of the medial superior olive corresponding to the tonotopic sequence in the posterior, but not the anterior, part of anterior AVCN. Possibly the projections of the anterior AVCN differ according to cell type rather than nuclear boundaries. Perhaps one cell type in anterior AVCN projects to the lateral superior olive, while another projects to the medial superior olive. The present results are consistent with bushy cell projections to the medial superior olive. If the granular labelling results from uptake at endings, then the projections to the superior olivary complex have a much wider origin in the cochlear nucleus than previously shown, including stellate cells in the ventral cochlear nucleus and certain neurons in the dorsal cochlear nucleus.

3049

third of the trapezoid body.30*31.36,4’ Degeneration studies show that the large ~lyciferous fibers originate in the central region of the contralateral ventral cochlear nucleus.‘5,‘4,35,47,48 The characteristic thickening of the bushy cell axons in the present material, seen also on globular cells with reduced silver methods,” is compatible with the possibility that the bushy (globular) cells form the calyces. Further evidence is supplied in the present material by following the tract of diffusely-filled, thick axon segments from AVCNp and the bushy (globular) cells through the middle-to-ventral third of the trapezoid body and then, on the other side of the injection, across the midline and into the contralateral MNT3. In the MNTB, many of the anterogradely-filled thick fibers form calyces. The marked propensity of the cellular elements with thick axons to fill heavily with HRP also suggests that calyces and bushy cells have the same fibers. A similar argument suggests that the thick fibers forming the dense collateral plexus in the ipsilateral lateral nucleus of the trapezoid body are also from the bushy cells. The HRP-filled plexus in the lateral trapezoid nucleus is identical with the plexus that Ram6n y Caja136 (Fig. 357) described as arising from thin trapezoid axons. Either there are two sources of the fibers which supply this nucleus, only one of which filled in the present material, or Ramcm y Cajal ascribed the collaterals to the wrong parent fibers. Elverland” apparently filled the same fibers anterogradely with HRP injected into the central region of the ventral cochlear nucleus and found them to ramify in the lateral peri-olivary region of the same side; he could not follow them across the midline to see whether they terminated in the MNTB.

Origin ofthe calyces qf Held

Physiological implications

HRP injections limited to the MNTB yield significant granular labelling of only the globular cell bodies in the contralateral AVCNp. This finding confirms the hy~thesis that globular cells in AVCNp project to the MNTB, as suggested on indirect grounds in the rat.15*‘”

Electrophysiological observations agree with the present conclusion that the cell type which receives end-bulbs from the cochlear nerve is the one that sends thick axons to the MNTB. Certain units in AVCNp, with a characteristic electrophysiological response pattern, exhibit small extracellular pre-potentials, thought to represent end-bulb activity; these units can be antidromically stimulated from the contralateral trapezoid body with very short laten-

Do bushy cells form the calyces on principal cells in the medial nucleus of the trapezoid body? The calyces

arise from the thickest fibers in the middle-to-ventral

Fig. 14.Cytological features of bushy and stellate cells. A. The bushy cell has few primary dendrites, each forming a spray of processes, or dendritic bush. The cell body often has appendages and contains free ribosomes. scattered cisterns of rough endoplasmic reticulum, a loose ring of Golgi apparatus, and an eccentric nucleus with a regular outline. Encrusting the perikaryal surface and proximal dendrites are numerous synaptic endings (only partially shown), including the large end-bulbs (I) from cochlear nerve fibers and smaller endings of types I, 2 and 3. The axon (a) has a thin tapering initial segment with a tortuous neck that expands to about 3 pm in diameter as the axon projects toward the trapezoid body without branching in the cochlear nucleus. B. The stellate cell is almost smooth and has a number of dendrites which taper gradually and branch infrequently. The cytoplasm of the larger neurons contains stacks of rough endoplasmic reticulum (Nissl bodies) and an invaginated, eucentric nucleus. Axonal endings, including a few type I synapses, tend to cluster at the basis of dendrites, while relatively few synapse on the cell body. The axon, thinner than that of the bushy cell, tapers gradually over its initial segment and may give off a collateral in the cochlear nucleus before entering the trapezoid body.

L. P. Tolbert.

3050

D. K. Morest

and D. A. Yurgeiun-Todd

Fig. 15. Functional neuroanatomy of the bushy cell in the region of the cochlear nerve root. Bushy cells in the posterior division of the anteroventral cochlear nucleus (AVCN) receive type 1. presumably excitatory, synapses from the fibers of spiral ganglion cells innervating hair cells in the cochlea. The bushy cells send thick, rapidly conducting axons to the contralateral medial nucleus of the trapezoid body (MNTB), where they form large axosomatic, excitatory calyces on the principal neurons. Precalycine collaterals go to the ipsilateral lateral nucleus of the trapezoid body (LNTB). The signal. or stimulus code, conveyed by cochlear nerve fibers, is represented by a primary type of peri-stimulus time (t) histogram. This signal is modified slightly by the bushy cells in AVCN to produce a notch in the histogram after the initial peak (pri-notch pattern). The calycine synapses in the MNTB are very secure and preserve the pri-notch pattern, which presumably would be altered in the LSO. The peri-stimulus time histograms correspond in general form to those previously reported.” Earlier authors have described related projections, indicated by dotted lines. The calycine axon also sends a oollaterai to the dorsomedial peri-olivary nucleus (DMPO). which contains crossed olivo-cochlear neurons (COCB). The principal neurons of the MNTB also project to the DMPO and to the lateral superior olive (LSO), a site of intensity-dependent binaural interactions. These additional pathways could be construed as parts of a recurrent circuit hypothesized to relate intensity coding to a laterahzation function (see text). MSO, medial superior olive.

cies4 indicating that they have thick axons which cross the midline. The sharp tuning curves and typical time-pattern of response (‘pri-notch’ pattern4) is preserved between the cochlcar nucleus and the MNTB.” This preservation can now be ascribed to the bushy cells of AVCNp and their calycine synapses with the principal cells of MNTB (Fig. 15). The MNTB principal neurons which receive calyces from AVCNp bushy cells send axons toward the lateral superior olive,30 and, indeed, degeneration,2.39 autoradiographic,” and HRP’.” methods substantiate such a projection (Fig. 15) although the precise synaptic relationships have not been demonstrated. The AVCNp-MNTB pathway may provide a fast route for contralateral input to those neurons which receive ipsilateral information from the anterior AVCN. Since the ipsilateral input to lateral superior olivary neurons can be excitatory when the contralateral input is inhibitory, 3,‘2,13 the olivary units. in a sense, compare the binaural inputs4’ In this way, the AVCNp, through its projection to the MNTB, may play a role in the lateralization of sounds according to

intensity differences. At the same time. the principal neurons of the MNTB and, presumably, the collaterals of the bushy cells of AVCNp project to the dorsomedial peri-olivary nucleus, which contains some of the crossed olivo-cochlear neurons28*3o that form an inhibitory pathway to the cochlea. Possibly then. these links in the pathway could provide a gainsetting feedback control of the at&rent system consistent with the optimum detection of intensity differences. Arknowlr~&~~ents This work was supported by USPHS grants 2R01 NS 06115. SROI NS 14347. 5TOl GM 00406, and IT01 MH 14275. We gratefully acknowledge Dr N. Cant’s contributions to the early phases of this work. A.P. Ley and 1. Downs provided excellent photographic assistance. We thank Drs J. Guinan. K. Osen, W. Brownell. J. Adams. and E. Kane for valuable discussions of various portions of this work. The principal findings were presented at the annual meeting of the society for Neuroscience on November 8. 197753 and were submitted by L. P. T. in partial fulfillment of the requirements for the Ph.D. degree from Harvard University.42

REFERENCES 1. Adams J. C. (1977) Technical 2, 141-14s.

considerations

on the use of horseradish

peroxidase

as a neuronal

marker.

R;ruroscirnce

Horseradish

peroxidase

in cochlear

nucleus

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2. Borg E. (1973) A neuroanatomical study of the brainstem auditory system of the rabbit-II. Descending connections. Acta morph. need-stand. 1 I, 49-62. 3. Boudreau J. C. & Tsuchitani C. (1968) Binaural interaction in the cat superior olive S-segment. J. Neurophysiol. 31. 442-454. 4. Bourk T. R. (1976) Electrical responses of neural units in the anteroventral cochlear nucleus of the cat. Doctorai dissertation. Massachusetts Institute of Technology, Cambridge. Massachusetts. 5. Brawer J. R. & Morest D. K. (1975) Relations between auditory nerve endings and cell types in the cat’s anteroventral cochlear nucleus seen with the Golgi method and Nomarski optics. J. camp. Neural. 160. 491-506. 6. Brawer J. R., Morest D. K. & Kane E. S. C. (1974) The neuronal architecture of the cochlear nucleus of the cat. J. camp. Neurol. 155, 251-300. 7. Brunso-Bechtold J., Glendenning K. K. & Masterton R. B. (1977) Some ascending projections of the medial nucleus of the trapezoid body in cat. Sot. Netcrosci. Ahs 3, 4. 8. Cant N. B. & Morest D. K. (1978) Axons from non-cochlear sources in the anteroventral cochlear nucleus of the cat. A study with the rapid Golgi method. Neuroscience 3, 100331029. 9. DeVito J. L.. Clausing K. W. & Smith 0. A. (1974) Uptake and transport of horseradish peroxidase by cut end of the vagus nerve. Bruin Res. 82. 269-271. 10. Elverland H. (1977) Descending connections between the superior olivary and cochlear nuclear complexes in the cat studied by autoradiographic and horseradish peroxidase methods. E.x$ Brrrin Res. 27, 3977412. il. Graham R. C.. Jr. & Karnovsky M. J. (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastru~tural cytochemistry by a new technique. J. Histochem. C~toc~lern. 14, 29 l-302. 12. Guinan J. J., Jr., Guinan S. S. & Norris B. E. (1972) Single auditory units in the superior olivary complex-I. Responses to sounds and classification based on physiological properties. Int. J. Nrurosci. 4, 101-120. 13. Guinan J. J.. Jr., Norris B. E. & Guinan S. S. (1972) Single auditory units in the superior olivary complex--II. Locations of unit categories and tonotopic organization. Inr. J. Nrurosci. 4. 147-166. 14. Halperin J. J. & LaVail J. H. (1975) A study of the dynamics of retrograde transport and accltmulation of horseradish peroxidase in injured neurons. Brain Rex 100, 253-269. 15. Harrison J. M. & Irving R. (1966) Ascending connections of the anterior ventral cochlear nucleus in the rat. J. camp. Neural. 126, 51-64. 16. Harrison J. M. & Irving R. (1966) The organization of the posterior ventral cochlear nucleus in the rat. J. camp. Neural. 126, 391-402. 17. Harrison J. M. & Warr W. B. (1962) A study of the cochlear nuclei and ascending auditory pathways of the medulla. J. camp. Neural. 119, 341-379. 18. Hedreen J. C. & McGrath S. (1977) Observations on iabelling of neuronal cell bodies, axons, and terminals after injection of horseradish peroxidase into rat brain. J. camp. Neurok 176, 225-246. 19. Holtzman E. & Peterson E. R. (1969) Uptake of protein by mammalian neurons. J. Cell Biol. 40, 863-869. 20. Kane E. S. C. (1976) Descending projections to specific regions of cat cochlear nucleus: a light microscopic study. Expl Neural. 52, 372-388. 21. Kane E. S. C. & Conlee J. W. (1979) Descending inputs to the caudal cochlear nucleus of the cat: degeneration and autoradiographi~ studies. J. camp. Netlrol. 187, 759-784. 22, Kiang N. Y. S., Morest D. K., Godfrey D. A., Guinan J. J., Jr. & Kane E. C. (1973) Stimulus coding at caudal levels of the cat’s auditory nervous system-I. Response characteristics of single units. In Basic Mechanisms in Hearing (ed. Moller A. R.) pp. 455-478. Academic Press, New York. 23. Kristensson K. & Olsson Y. (1973) Uptake and retrograde transport of protein tracers in hypoglossal neurons. Fate of the tracer and reaction of the nerve cell bodies. Acta neuropathol. (Bed.) 23, 43-47. 24. Kristensson K. & Olsson Y. (1974) Retrograde transport of horseradish peroxidase in transected axons-I. Time relationships between transport and induction of chromatolysis. Brain RPS. 79, 101-109. 25. LaVail J. H. 81 LaVail M. M. (1974) The retrograde intraaxonal transport of horseradish proxidase in the chick visual system: a light- and electron-microscopic study. J. camp. Neural. 157, 303-358. 26. Lorente de No R. (1976) Some unresolved problems concerning the cochlear nerve. Ann. Otol. Rhino/. Lar. 85 (Suppl. 34) I-28. 27. Lorente de No (1981) The Primary Acoustic Nuclei New York, Raven. 28. Luk G. D., Morest D. K. & McKenna N. M. (1974) Origins of the crossed olivocochlear bundles shown by an acid phosphatase method in the cat. Ann. Otol Rh~nol. Lar. 83, 382-392. 29. Lund J. S.. Lund R. D., Hendrickson A. E., Bunt A. B. & Fuchs A. F. (1975) The origin of efferent pathways from the primary visual cortex, area 17, of the macaque monkey as shown by retrograde transport of horseradish peroxidase. J. camp. Neurol. 164, 287-304. 30. Morest D. K. (1968) The collateral system of the media1 nucleus of the trapezoid body of the cat, its neuronal architecture and relation to the olivocochlear bundle. Brain Res. 9, 288-31 I. 31. Morest D. K. (1968) The growth of synaptic endings in the mammalian brain: a study of the calyces of the trapezoid body. 2. Anat. E~t~~Gesch. 127, 201-220. 32. Oliver D. L. & Hail W. C. (1978) The medial geniculate body of the tree shrew, Tupaia g&-I. Cytoarchitecture and midbrain connections, J. camp. Neural. 182, 423-458. 33. Osen K. K. (1969) Cytoarchitecture of the cochlear nuclei in the cat. J. camp. Neural. 136, 453-484. 34. Osen K. K. (1969) The intrinsic organization of the cochlear nuclei in the cat. Acta oto-laryngol 67, 352-359. 35. Poljak S. (1926) The connections of the acoustic nerve. J. Anat. 60, 465-469.

3052

L. P. Tolbert.

D. K. Morest

and D. A. Yurgelun-Todd

36. Ramon y Cajal S. (1909) Histologic du SystPme Nrrurux de PHomme et des VertPhrGs Vol. 1 (1952 reprint). pp. 754838. Instituto Ramon y Cajal, Madrid. 37. Rasmussen G. L. (1960) E&rent fibers of the cochlear nerve and cochlear nucleus. In Neural Mechanisms of the Auditory and Vestibular Systems (eds Rasmussen G. L. & Windle W. F.) pp. 105-115. Thomas, Springfield. 38. Rasmussen G. L. (1964) Anatomic relationships of the ascending and descending auditory systems. In Neurological Aspects qf Auditory and Vestibular Disorders (eds Fields W. S. & Alford B. R.) pp. 5523. Thomas, Springfield. 39. Rasmussen G. L. (1967) Efferent connections of cochlear nucleus. In Sensorineural Hearing Processes and Disorders (ed. Graham A. B.) pp. 61-75. Little, Brown, Boston. 40. Rasmussen G. L., Gacek R. R., McCrane E. P. & Baker C. C. (1960) Model of cochlear nucleus (cat) displaying its aRerent and etTerent connections. Anat. Rec. 136, 344. 41. Statler W. A. (1953) An experimental study of the cells and connections of the superior olivary complex of the cat. J. cornp. Neural. 98, 401423. 42. Tolbert L. P. (1978) Synaptic organization in the anteroventral cochlear nucleus of the cat: a light- and electronmicroscope study. Doctoral dissertation, Harvard University, Cambridge, Massachusetts. 43. Tolbert L. P. & Morest D. K. (1977) Combined Golgi, horseradish peroxidase (HRP). and electron-microscopic study of bushy cells in the cochlear nucleus. Sot. Neurosci. Abs 3. 12. 44. Tolbert L. P. & Morest D. K. (1982) The neuronal architecture of the anteroventral cochlear nucleus of the cat in the region of the cochlear nerve root: Golgi and Nissl methods. Neuroscience 7, 3013-3030. 45. Tolbert L. P. & Morest D. K. (1982) The neuronal architecture of the anteroventral cochlear nucleus of the cat in the region of the cochlear nerve root: electron-microscopy. Neuroscience 7, 305333067. 46. Tsuchitani C. & Boudreau J. (1969) Stimulus level of dichotically presented tones and cat superior olive S-segment cell discharge. J. acoust. Sot. Am. 46, 979-988. 47. van Noort J. (1969) The Structure and Connections of the Inferior Colliculus. An Inuestigution of fhe Lower Auditor) System. Van Gorcum, Assen. 48. Warr W. B. (1972) Fiber degeneration following lesions in the multipolar and globular cell areas in the ventral cochlear nucleus of the cat. Brain Res. 40, 2477270. (Accepted

21 May 1982)